The C-Type Lectin of the Aggrecan G3 Domain Activates Complement

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint

Versican G3 Promotes Mouse Mammary Tumor Cell Growth, Migration, and Metastasis by Influencing EGF Receptor Signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis

ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression

SummaryWe show that combinatorial mouse alleles for the secreted metalloproteases Adamts5, Adamts20 (bt), and Adamts9 result in fully penetrant soft-tissue syndactyly. Interdigital webs in Adamts5−/−;bt/bt mice had reduced apoptosis and decreased cleavage of the proteoglycan versican; however, the BMP-FGF axis, which regulates interdigital apoptosis was unaffected. BMP4 induced apoptosis, but without concomitant versican proteolysis. Haploinsufficiency of either Vcan or Fbln1, a cofactor for versican processing by ADAMTS5, led to highly penetrant syndactyly in bt mice, suggesting that cleaved versican was essential for web regression. The local application of an aminoterminal versican fragment corresponding to ADAMTS-processed versican, induced cell death in Adamts5−/−;bt/bt webs. Thus, ADAMTS proteases cooperatively maintain versican proteolysis above a required threshold to create a permissive environment for apoptosis. The data highlight the developmental significance of proteolytic action on the ECM, not only as a clearance mechanism, but also as a means to generate bioactive versican fragments

Methods for Monitoring Matrix-Induced Autophagy.

A growing body of research demonstrates modulation of autophagy by a variety of matrix constituents, including decorin, endorepellin, and endostatin. These matrix proteins are both pro-autophagic and anti-angiogenic. Here, we detail a series of methods to monitor matrix-induced autophagy and its concurrent effects on angiogenesis. We first discuss cloning and purifying proteoglycan fragment and core proteins in the laboratory and review relevant techniques spanning from cell culture to treatment with these purified proteoglycans in vitro and ex vivo. Further, we cover protocols in monitoring autophagic progression via morphological and microscopic characterization, biochemical western blot analysis, and signaling pathway investigation. Downstream angiogenic effects using in vivo approaches are then discussed using wild-type mice and the GFP-LC3 transgenic mouse model. Finally, we explore matrix-induced mitophagy via monitoring changes in mitochondrial DNA and permeability

Effects of low-dose X-irradiation on mouse-brain aggregation cultures

Biochemical and morphological differentiation in reaggregating mouse-brain cell cultures after low-dose radiation (0.5 Gy) in vitro was studied. Cells were irradiated on culture day 2, corresponding to embryonic day 15-16, and different glial and neuronal markers were followed through development to postnatal day 40. The shape and size of irradiated aggregates were more irregular and smaller compared with controls. Total amounts of DNA and protein were significantly lower in irradiated aggregates than in controls between days 8 and 20. After 30 days in culture activities of the glial markers glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were lower in X-irradiated aggregates than in controls. However, after 40 days the CNP activity in irradiated aggregates increased to levels above those of the controls. Irradiated and control aggregates did not differ significantly in neuronal marker enzyme activities, i.e. choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and glutamic acid decarboxylase (GAD) measured on a per mg protein basis. On days 20 and 30 the amount of nerve growth factor (NGF) was two-fold higher in irradiated aggregates compared with non-irradiated ones, suggesting that, after irradiation, surviving cells in culture were induced to produce more NGF. After 40 days the amount of NGF in irradiated aggregates had decreased to the level found in the control aggregates

Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin